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WO2018159841A1 - Novel compound, fluorescence derivatization reagent including said novel compound, method for optically resolving optical isomer of amino acid in which said novel compound is used, and fluorescence derivatized amino acid - Google Patents

Novel compound, fluorescence derivatization reagent including said novel compound, method for optically resolving optical isomer of amino acid in which said novel compound is used, and fluorescence derivatized amino acid Download PDF

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WO2018159841A1
WO2018159841A1 PCT/JP2018/008154 JP2018008154W WO2018159841A1 WO 2018159841 A1 WO2018159841 A1 WO 2018159841A1 JP 2018008154 W JP2018008154 W JP 2018008154W WO 2018159841 A1 WO2018159841 A1 WO 2018159841A1
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amino acid
novel compound
fluorescence
compound
amino acids
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French (fr)
Japanese (ja)
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健司 浜瀬
健行 秋田
真史 三田
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Shiseido Co Ltd
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Shiseido Co Ltd
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Priority to AU2018228242A priority Critical patent/AU2018228242A1/en
Priority to EP18760371.7A priority patent/EP3590937A4/en
Priority to CN201880012088.5A priority patent/CN110366552A/en
Priority to SG11201908102T priority patent/SG11201908102TA/en
Priority to JP2019503161A priority patent/JPWO2018159841A1/en
Priority to US16/490,713 priority patent/US20200002322A1/en
Publication of WO2018159841A1 publication Critical patent/WO2018159841A1/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/12—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B51/00—Nitro or nitroso dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
    • C09B67/0071—Process features in the making of dyestuff preparations; Dehydrating agents; Dispersing agents; Dustfree compositions
    • C09B67/0083—Solutions of dyes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8818—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving amino acids

Definitions

  • the present invention provides a novel compound, a fluorescent derivatization reagent containing the novel compound, a method for optical resolution of a fluorescent derivatized amino acid obtained by reacting the novel compound with an amino acid, and a reaction of the novel compound with an amino acid. And fluorescent derivatives of amino acids.
  • Non-Patent Documents 1-3 D-amino acids and L-amino acids are precisely used using high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • analysis methods for separating and quantifying for example, see Non-Patent Document 4 are underway.
  • fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) a conventional reagent for HPLC labeling, is combined with two-dimensional chiral HPLC.
  • NBD-F 4-fluoro-7-nitro-2,1,3-benzoxadiazole
  • a method for selectively analyzing all amino acids constituting a protein has been developed (Non-patent Document 5).
  • a reagent for optical resolution capable of optically resolving a mixture of optical isomers of amino acids with higher sensitivity and ease has been developed (Patent Document 2).
  • NBD-F 4-fluoro-7-nitro-2,1,3-benzooxadiazole
  • the present invention thus relates to: [1] The following formula: Or a salt thereof.
  • a reagent for fluorescent derivatization comprising a compound represented by the formula: [3] A method for analyzing an amino group-containing compound in a sample, A sample containing the amino group-containing compound and the following formula: Reacting with a compound represented by: Introducing the reactant into chromatography.
  • the analytical method is a method of optical resolution of optical isomers, and the chromatography is chiral chromatography.
  • the method according to item 3 or 4 wherein the amino group-containing compound is an amino acid.
  • a decrease in detection sensitivity due to quenching can be suppressed, and a compound for fluorescent derivatization used for optical resolution can be provided.
  • FIG. 1 relates to a chromatogram obtained by subjecting a sample containing protein-constituting amino acids to fluorescence derivatization with NBD-F and subjecting it to HPLC analysis.
  • FIG. 2 shows that a sample containing D-alanine and L-alanine and D-tryptophan and L-tryptophan was derivatized with NBD-F (A) or NBD-COOSu (B) and (C) to obtain chiral HPLC.
  • a chromatogram obtained by analysis is shown. In (A) and (B), the chiral HPLC separation was measured by fluorescence analysis, while in (C) the chiral HPLC separation was measured by mass spectrometry.
  • one aspect of the present invention is a compound of the following formula: Or a salt thereof. Salts can include salts formed with any acid.
  • inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate and perchlorate, or acetate, oxalate, maleate, tartrate, citrate, succinate
  • organic acid salts such as benzoate or malonate.
  • the compound of the present invention emits fluorescence based on nitrobenzooxadiazole (NBD) and reacts with a compound having an amino group to generate a fluorescent derivative based on the reactivity of the succinimidyl group.
  • NBD nitrobenzooxadiazole
  • Any compound having an amino group can react with any compound, and examples thereof include amino acids.
  • a fluorescent derivative of an amino acid is generated based on the following scheme.
  • NBD-COOSu Fluorescent derivatization reagent containing -methylamino] acetate
  • D-amino acids and L-amino acids can be separated by introducing an amino acid that has been fluorescently derivatized with a fluorescent derivatizing reagent into chiral column chromatography. It can also be referred to as a reagent for resolution.
  • R represents an arbitrary side chain of an amino acid
  • the present invention also relates to a method for producing a fluorescent derivative of an amino acid comprising reacting the compound of the present invention with an amino acid.
  • a fluorescent derivative of an amino acid produced by the production method that is, the following formula: (In the formula, R represents a side chain of any amino acid) It also relates to amino acid fluorescent derivatives having In the present invention, R represents a side chain of any amino acid, but may be a side chain of a protein-constituting amino acid. Further, from the viewpoint of optical resolution, it is preferably an arbitrary side chain other than the glycine side chain (-H) among the amino acids constituting the protein.
  • amino acid fluorescent derivatives are characterized in that they can emit fluorescence due to the NBD moiety and do not cause fluorescence quenching that may occur due to the structure of some amino acid side chains. Further, these amino acid fluorescent derivatives retain the chirality of the original amino acid. Therefore, it is also a feature that D-amino acid and L-amino acid can be separated based on the chirality of the original amino acid by introducing an amino acid fluorescent derivative into chiral column chromatography.
  • an optical resolution reagent containing the compound of the present invention may be related to a kit or system for optical resolution and / or fluorescence derivatization including an optical resolution and / or fluorescence derivatization reagent and an optical resolution column.
  • the present invention relates to a method for analyzing an amino group-containing compound in a sample.
  • a sample containing an amino group-containing compound and the following formula: Reacting with a compound represented by: Introducing the reactant into the chromatography.
  • the analysis method of the present invention is particularly preferably an optical resolution method for separating optical isomers. In this case, chiral chromatography is performed as chromatography. Any compound having an amino group can be used.
  • amino acids can be mentioned, and optical isomers of amino acids, particularly protein-constituting amino acids, that is, D-amino acids and L-amino acids can be separated and analyzed.
  • the step of reacting the sample containing the amino group-containing compound with the compound of the present invention comprises reacting the sample containing the amino group-containing compound with the solution containing the compound of the present invention and optionally heating. Can do. This reaction is preferably performed in the dark for the purpose of avoiding fluorescent fading.
  • a suitable solvent can be arbitrarily used, for example, water, methanol, ethanol, acetonitrile and the like can be used.
  • the sample containing the amino group-containing compound may be any sample such as a biological sample, an environmental sample, an experimental sample, and an industrial sample (food sample, pharmaceutical sample, cosmetic sample).
  • Biological samples include body fluids such as blood, saliva, tears and lymph, tissue samples such as cells and organs, and excrement samples such as urine and feces. These samples may be directly reacted with the compound of the present invention, but it is preferable to purify appropriately according to the sample before the reaction step. Further, purification can be performed after the reaction step. Purification of the sample can be performed by appropriately selecting or combining filtration, slow protein, solvent extraction, ion exchange, solid phase extraction, and the like according to the type of sample.
  • the amino group-containing compound that has been derivatized by reacting with the compound of the present invention is subjected to chromatography equipped with an optical resolution column.
  • chromatography HPLC is preferred.
  • chromatography may be performed in advance using another column system.
  • the optical resolution column a column capable of separating D-amino acid and L-amino acid derivatized with the compound of the present invention can be arbitrarily selected.
  • the optical resolution column uses a compound containing only one optical isomer as a stationary phase. For example, a glycine derivative, a leucine derivative, a quinine derivative, or the like can be used.
  • the amino acid derivative separated into D-amino acid and L-amino acid by chromatography can be analyzed by detecting the fluorescence.
  • the detection of fluorescence can be performed in a detector included in the chromatography system.
  • the detector can analyze the derivatized amino acid by irradiating excitation light (470 nm) for exciting NBD and detecting fluorescence (fluorescence wavelength: 530 nm) derived from the excited NBD.
  • Protein-constituting amino acids refer to 20 types of amino acids that constitute proteins.
  • As protein constituting amino acids alanine (Ala / A), arginine (Arg / R), asparagine (Asn / N), aspartic acid (Asp / D), cysteine (Cys / C), glutamic acid (Glu / E), glutamine (Gln / Q), glycine (Gly / G), histidine (His / H), isoleucine (Ile / I), leucine (Leu / L), lysine (Lys / K), methionine (Met / M), phenylalanine ( Phe / F), proline (Pro / P), serine (Ser / S), threonine (Thr / T), tryptophan (Trp / W), tyrosine (Tyr / Y), and valine (Val / V).
  • amino acids to be analyzed are preferably protein-constituting amino acids excluding glycine, but other amino acids such as ornithine and citrulline can also be analyzed. it can.
  • Example 1 2,5-Dioxopyrrolidin-1-yl 2- [N- (4-nitrobenzo [c] [1,2,5] oxadiazol-7-yl) -N-methylamino] acetate ( Synthesis of NBD-COOSu) (1) Synthesis of NBD-COOH In the dark, N-methylglycine (1.85 g, 20.8 mmol) was dissolved in 0.52 M aqueous sodium hydrogen carbonate solution (100 mL), and NBD-Cl (1.04 g, 5.23 mmol) in acetonitrile was dissolved in the solution. (50 mL) was added dropwise. After stirring the solution at room temperature for 2 hours, acetonitrile was distilled off under reduced pressure.
  • NBD-COOSu 2,5-Dioxopyrrolidin-1-yl 2- [N- (4-nitrobenzo [c] [1,2,5] oxadiazol-7-yl) -N-methylamino] acetate
  • NBD-COOH prepared in Example 1 (236 mg, 0.935 mmol) was dissolved in methylene chloride (2 mL), and oxalyl chloride (1.1 mL) was added dropwise to the solution.
  • N, N-dimethylformamide (5 ⁇ L) was added to the solution and stirred at room temperature for 1 hour. Thereafter, the solvent was distilled off under reduced pressure, and vacuum drying was performed.
  • Example 2 Fluorescence derivatization of amino acids with NBD-COOSu 400 mM borate buffer solution (10 ⁇ L) containing 20 pM D-tryptophan and 80 pM L-tryptophan respectively, and 400 mM borate buffer solution (10 ⁇ L) containing 0.5 pM D-alanine and 2 pM L-alanine respectively (10 ⁇ L) 20 ⁇ L of pH 8.0) was added, and 40 mM NBD-COOSu in acetonitrile (5 ⁇ L) was added, followed by reaction at 60 ° C. for 2 minutes. Next, 0.2% aqueous trifluoroacetic acid solution (75 ⁇ L) was added to stop the reaction, and amino acid derivatives (NBD-CO-Trp and NBD-CO-Ala) were obtained.
  • 400 mM borate buffer solution (10 ⁇ L) containing 20 pM D-tryptophan and 80 pM L-tryptophan respectively 400 mM borate buffer solution (10
  • Example 3 Separation by HPLC
  • the amino acid derivative obtained in Example 2 was subjected to HPLC analysis.
  • the column used for the HPLC analysis is an original Prickle type column (Patent No. 597765) using KSAACSP-001R (1.5 mm id ⁇ 250 mm) as a chiral stationary phase, and acetonitrile: methanol (30:70) as a mobile phase.
  • Detection was performed using a fluorescence (excitation wavelength: 470 nm, fluorescence wavelength: 530 nm) and UV / VIS (measurement wavelength: 470 nm) detector.
  • the aqueous solution of alanine derivative was subjected to HPLC analysis.
  • the column was washed (methanol 10 minutes, ammonium formate 10 minutes, and methanol 20 minutes), and the eluate was stabilized for 40 minutes, and then the tryptophan derivative aqueous solution was subjected to HPLC analysis.
  • the chromatogram obtained by fluorescence detection is shown in FIG. 2B. Further, instead of fluorescence detection, detection was performed by mass spectrometry. The chromatogram detected by mass spectrometry is shown in FIG. 2C.
  • Comparative Example 1 HPLC analysis using NBD-F 10 ⁇ L of 400 mM borate buffer (pH 8.0) was added to a standard sample of amino acids containing protein-constituting amino acids and allobodies, and a 40 mM NBD-F solution ( 5 ⁇ L) and reacted at 60 ° C. for 2 minutes. Next, 0.2% trifluoroacetic acid aqueous solution (75 ⁇ L) was added to stop the reaction to obtain an amino acid derivative. The obtained amino acid derivative was subjected to HPLC equipped with a reverse phase column (monolithic ODS column, 0.53 mm id ⁇ 1000 mm; Shiseido) set at 45 ° C.
  • a reverse phase column monolithic ODS column, 0.53 mm id ⁇ 1000 mm; Shiseido
  • Comparative Example 2 Chiral HPLC analysis using NBD-F.
  • 10 ⁇ L of 400 mM borate buffer (pH 8.0) was added to each alanine aqueous solution (10 ⁇ L), 40 mM NBD-F solution (5 ⁇ L) was further added, and the mixture was reacted at 60 ° C. for 2 minutes.
  • Example 3 the results of derivatizing alanine and tryptophan using NBD-COOSu and subjecting each derivative to chiral HPLC were shown. For both alanine and tryptophan, D-form and L-form peaks were observed separately (FIG. 2B). This peak coincided with the result detected by mass spectrometry. From these results, it was shown that derivatization of amino acids using NBD-COOSu is effective for separating and detecting amino acids including D-form and L-form.

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Abstract

The purpose of the present invention is to develop an optical resolution reagent for which quenching does not occur in the analysis of a chiral amino acid. The problem is solved by providing a novel compound for optical resolution that does not cause quenching. The present invention pertains to a novel compound, an optical resolution reagent that includes the novel compound, an optical resolution method that includes a step for reacting the novel compound, and an optical isomer obtained by reacting the novel compound with an amino acid.

Description

新規化合物、当該新規化合物を含む蛍光誘導体化用試薬、並びに当該新規化合物を用いたアミノ酸の光学異性体を光学分割する方法及び蛍光誘導体化されたアミノ酸Novel compound, fluorescent derivatization reagent containing the novel compound, method for optical resolution of optical isomers of amino acids using the novel compound, and fluorescent derivatized amino acid

　本発明は新規化合物、当該新規化合物を含む蛍光誘導体化用試薬、及び当該新規化合物をアミノ酸と反応させて得た蛍光誘導体化アミノ酸を光学分割する方法、及び当該新規化合物をアミノ酸と反応させて得たアミノ酸の蛍光誘導体に関する。 The present invention provides a novel compound, a fluorescent derivatization reagent containing the novel compound, a method for optical resolution of a fluorescent derivatized amino acid obtained by reacting the novel compound with an amino acid, and a reaction of the novel compound with an amino acid. And fluorescent derivatives of amino acids.

　従来、高等動物の生体中に存在するアミノ酸は、Ｌ－アミノ酸のみであると考えられてきた。しかしながら、近年、分析技術の発展により、高等動物、特にヒトを含む哺乳類の生体内にもＤ－アミノ酸が存在し、記憶・学習やホルモン産生・分泌などの生理機能を有することが明らかになっている。また、様々な組織や生体試料において存在するＤ－アミノ酸のうちの幾つかが、体の状態、例えば腎不全などの疾患により変動することが示されており、健康・疾患マーカーとしての利用が期待されている（特許文献１）。 Conventionally, it has been considered that the only amino acid present in the living body of higher animals is L-amino acid. However, in recent years, with the development of analytical techniques, it has become clear that D-amino acids also exist in the living body of higher animals, particularly mammals including humans, and have physiological functions such as memory / learning and hormone production / secretion. Yes. In addition, some of D-amino acids present in various tissues and biological samples have been shown to vary depending on the body condition, for example, diseases such as renal failure, and are expected to be used as health and disease markers. (Patent Document 1).

　生体内のアミノ酸の生理的役割を解明する観点から、高速液体クロマトグラフィー（ＨＰＬＣ）法を利用して、アミノ酸（例えば、非特許文献１－３参照）やＤ－アミノ酸及びＬ－アミノ酸を精密に分離して定量する分析手法（例えば、非特許文献４参照）の開発研究が進められている。また、従来のＨＰＬＣラベル化用の試薬である４－フルオロ-７-ニトロ-２，１，３-ベンゾオキサジアゾール（ＮＢＤ－Ｆ）により蛍光誘導体化を図り、２次元キラルＨＰＬＣを組み合わせることで、タンパク質を構成するアミノ酸の全てを選択的に分析する方法が開発されている（非特許文献５）。さらに、より高感度で簡便にアミノ酸の光学異性体混合物を光学分割できる光学分割用の試薬も開発されている（特許文献２）。 From the viewpoint of elucidating the physiological role of amino acids in living organisms, amino acids (see, for example, Non-Patent Documents 1-3), D-amino acids and L-amino acids are precisely used using high performance liquid chromatography (HPLC). Research and development of analysis methods for separating and quantifying (for example, see Non-Patent Document 4) are underway. In addition, fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), a conventional reagent for HPLC labeling, is combined with two-dimensional chiral HPLC. A method for selectively analyzing all amino acids constituting a protein has been developed (Non-patent Document 5). Furthermore, a reagent for optical resolution capable of optically resolving a mixture of optical isomers of amino acids with higher sensitivity and ease has been developed (Patent Document 2).

国際公開第２０１３／１４０７８５号公報International Publication No. 2013/140785 特開２０１５－４８３３２号公報Japanese Patent Laying-Open No. 2015-48332

K．Shimbo et al., Anal．Chem．，2009，81，5172－5179K. Shimbo et al., Anal. Chem. , 2009, 81, 5172-5179 K．Shimbo et al., Rapid　Commun．Mass　Spectrom．，2009，23，1483－1492K. Shimbo et al., Rapid Commun. Mass Spectrom. , 2009, 23, 1483-1492 S．A．Cohen et al.,　Anal．Biochem．，1993，211，279－287S. A. Cohen etal., Anal. Biochem. , 1993, 211, 279-287 R．J．Reischl et al., J．Chromatogra．A．，2012，1269，262－269R. J. Reischl et al., J. Chromatogra. A. , 2012, 1269, 262-269 T. Kimura, et al., Scientific Reports, (2016), 6, 26137T. Kimura, et al., Scientific Reports, (2016), 6, 26137

　従来のＨＰＬＣラベル化用の試薬である４－フルオロ-７-ニトロ-２，１，３-ベンゾオキサジアゾール（ＮＢＤ－Ｆ）を用いたＤ－アミノ酸の分析技術では、トリプトファンとＮＢＤ－Ｆ試薬が反応して生じるＮＢＤ－トリプトファンにおいて、消光が生じてしまい、検出感度が低くなるという課題を見出した。一般的なアミノ酸分析システムの測定レンジは３～４桁オーダーであるが、ＮＢＤ－トリプトファンは消光により測定値がこのレンジに収まらず、４０以上ある他のキラルアミノ酸との同時分析が困難となるため、代謝物を網羅的に分析するメタボロミクスの手法に適用できないことがあった。 Conventional D-amino acid analysis technology using 4-fluoro-7-nitro-2,1,3-benzooxadiazole (NBD-F), which is a reagent for HPLC labeling, uses tryptophan and NBD-F reagent. In the NBD-tryptophan produced by the reaction, quenching occurs and the detection sensitivity is lowered. The measurement range of a general amino acid analysis system is on the order of 3 to 4 digits, but NBD-tryptophan does not fit in this range due to quenching, and simultaneous analysis with 40 other chiral amino acids becomes difficult. In some cases, it could not be applied to metabolomics methods for comprehensive analysis of metabolites.

　上記課題に対して、アミノ酸を誘導体化した際に、消光が生じない化合物について探索したところ、以下の式：

Figure JPOXMLDOC01-appb-C000006
　で表される化合物でトリプトファンを誘導体化した場合に、得られたトリプトファン誘導体について消光が生じず、またトリプトファン誘導体のＤ－アミノ酸とＬ－アミノ酸をキラルカラムクロマトグラフィーによって分離可能であることを見いだし、本発明に至った。 In response to the above problem, when a compound that does not quench when an amino acid is derivatized, the following formula:
Figure JPOXMLDOC01-appb-C000006
 It was found that when tryptophan was derivatized with a compound represented by the formula, quenching did not occur for the obtained tryptophan derivative, and D-amino acid and L-amino acid of the tryptophan derivative could be separated by chiral column chromatography. Invented.

　そこで本発明は以下に関する：
[１]　以下の式：

Figure JPOXMLDOC01-appb-C000007
　で表される化合物、又はその塩。
[２]　以下の式：
Figure JPOXMLDOC01-appb-C000008
 　で表される化合物、又はその塩を含む、蛍光誘導体化用試薬。
[３]　試料中のアミノ基含有化合物を分析する方法であって、
　当該アミノ基含有化合物を含む試料と、以下の式：
Figure JPOXMLDOC01-appb-C000009
 　で表される化合物とを反応させる工程；
　反応物をクロマトグラフィーに導入する工程
　を含む、前記方法。
[４]　前記分析方法が、光学異性体を光学分割する方法であり、クロマトグラフィーがキラルクロマトグラフィーである、項目３に記載の方法。
[５]　前記アミノ基含有化合物が、アミノ酸である、項目３又は４に記載の方法。
[６]　アミノ酸の蛍光誘導体の製造方法であって、
　アミノ酸と、以下の式：
Figure JPOXMLDOC01-appb-C000010
 　で表される化合物とを反応させることを含む、前記方法。
[７]　以下の式：
Figure JPOXMLDOC01-appb-C000011
 [式中、Ｒは、アミノ酸の側鎖である]
　で表される化合物。 The present invention thus relates to:
[1] The following formula:
Figure JPOXMLDOC01-appb-C000007
 Or a salt thereof.
[2] The following formula:
Figure JPOXMLDOC01-appb-C000008
 A reagent for fluorescent derivatization, comprising a compound represented by the formula:
[3] A method for analyzing an amino group-containing compound in a sample,
A sample containing the amino group-containing compound and the following formula:
Figure JPOXMLDOC01-appb-C000009
 Reacting with a compound represented by:
Introducing the reactant into chromatography.
[4] The method according to item 3, wherein the analytical method is a method of optical resolution of optical isomers, and the chromatography is chiral chromatography.
[5] The method according to item 3 or 4, wherein the amino group-containing compound is an amino acid.
[6] A method for producing a fluorescent derivative of an amino acid,
Amino acids and the following formula:
Figure JPOXMLDOC01-appb-C000010
 Reacting with a compound represented by the formula:
[7] The following formula:
Figure JPOXMLDOC01-appb-C000011
 [Wherein R is an amino acid side chain]
A compound represented by

　本発明によれば、消光による検出感度の低下を抑えることができ、光学分割に用いられる蛍光誘導体化用の化合物を提供できる。 According to the present invention, a decrease in detection sensitivity due to quenching can be suppressed, and a compound for fluorescent derivatization used for optical resolution can be provided.

図１は、タンパク質構成アミノ酸を含有する試料を、ＮＢＤ－Ｆで蛍光誘導体化して、ＨＰＬＣ分析に供して得たクロマトグラムに関する。FIG. 1 relates to a chromatogram obtained by subjecting a sample containing protein-constituting amino acids to fluorescence derivatization with NBD-F and subjecting it to HPLC analysis. 図２は、Ｄ－アラニン及びＬ－アラニン、及びＤ－トリプトファン及びＬ－トリプトファンを含有する試料を、ＮＢＤ－Ｆ（Ａ）又はＮＢＤ－ＣＯＯＳｕ（Ｂ）及び（Ｃ）で誘導体化して、キラルＨＰＬＣ分析に供して得たクロマトグラムを表す。（Ａ）及び（Ｂ）では、キラルＨＰＬＣの分離物を蛍光分析により測定している一方で、（Ｃ）ではキラルＨＰＬＣの分離物を質量分析により測定した。FIG. 2 shows that a sample containing D-alanine and L-alanine and D-tryptophan and L-tryptophan was derivatized with NBD-F (A) or NBD-COOSu (B) and (C) to obtain chiral HPLC. A chromatogram obtained by analysis is shown. In (A) and (B), the chiral HPLC separation was measured by fluorescence analysis, while in (C) the chiral HPLC separation was measured by mass spectrometry.

（新規化合物）
　本発明者らは、下記式で表される化合物(２，５－ジオキソピロリジン－１－イル２－[Ｎ‐（４－ニトロベンゾ[ｃ][１，２，５]オキサジアゾール－７－イル）‐Ｎ‐メチルアミノ］アセテート（ＮＢＤ－ＣＯＯＳｕ）)を新規に製造した。したがって、本発明の一の態様は、以下の式：

Figure JPOXMLDOC01-appb-C000012
で表される化合物又はその塩に関する。塩としては、任意の酸と形成された塩を挙げることができる。例えば塩酸塩、臭化水素酸塩、リン酸塩、硫酸塩及び過塩素酸塩などの無機酸塩、又は酢酸塩、シュウ酸塩、マレイン酸塩、酒石酸塩、クエン酸塩、コハク酸塩、安息香酸塩又はマロン酸塩などの有機酸塩が挙げられる。 (New compound)
The present inventors have identified a compound represented by the following formula (2,5-dioxopyrrolidin-1-yl 2- [N- (4-nitrobenzo [c] [1,2,5] oxadiazole-7- Yl) -N-methylamino] acetate (NBD-COOSu)). Accordingly, one aspect of the present invention is a compound of the following formula:
Figure JPOXMLDOC01-appb-C000012
 Or a salt thereof. Salts can include salts formed with any acid. For example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate and perchlorate, or acetate, oxalate, maleate, tartrate, citrate, succinate, Examples include organic acid salts such as benzoate or malonate.

（新規化合物の合成）
　この化合物は、任意の方法により製造されてもよいが、例えば下記のスキーム１及びスキーム２に基づいて製造することができる。

Figure JPOXMLDOC01-appb-C000013
(Synthesis of new compounds)
Although this compound may be manufactured by arbitrary methods, it can be manufactured based on the following scheme 1 and scheme 2, for example.
Figure JPOXMLDOC01-appb-C000013

（蛍光誘導体化用試薬及びその反応）
　本発明の化合物は、ニトロベンゾオキサジアゾール（ＮＢＤ）に基づいて蛍光を発すると共に、スクシンイミジル基の反応性に基づいて、アミノ基を有する化合物と反応して蛍光誘導体を生成する。アミノ基を有する化合物であれば、任意の化合物と反応することができ、例えばアミノ酸が挙げられる。具体的に、例えば下記のスキームに基づき、アミノ酸の蛍光誘導体を生成する。したがって、本発明の別の態様では、２，５－ジオキソピロリジン―１－イル２－[Ｎ‐（４－ニトロベンゾ[ｃ][１，２，５]オキサジアゾール－７－イル）‐Ｎ‐メチルアミノ]アセテート（ＮＢＤ－ＣＯＯＳｕ）を含む蛍光誘導体化用試薬に関する。後述のとおり、蛍光誘導体化試薬により蛍光誘導体化されたアミノ酸をキラルカラムクロマトグラフィーに導入することで、Ｄ－アミノ酸とＬ－アミノ酸を分離することができることから、本発明の蛍光誘導体化試薬は、光学分割用試薬ということもできる。

Figure JPOXMLDOC01-appb-C000014
（式中、Ｒは、アミノ酸の任意の側鎖を表す） (Fluorescent derivatization reagent and its reaction)
The compound of the present invention emits fluorescence based on nitrobenzooxadiazole (NBD) and reacts with a compound having an amino group to generate a fluorescent derivative based on the reactivity of the succinimidyl group. Any compound having an amino group can react with any compound, and examples thereof include amino acids. Specifically, for example, a fluorescent derivative of an amino acid is generated based on the following scheme. Thus, in another aspect of the invention, 2,5-dioxopyrrolidin-1-yl 2- [N- (4-nitrobenzo [c] [1,2,5] oxadiazol-7-yl) -N Fluorescent derivatization reagent containing -methylamino] acetate (NBD-COOSu). As described later, D-amino acids and L-amino acids can be separated by introducing an amino acid that has been fluorescently derivatized with a fluorescent derivatizing reagent into chiral column chromatography. It can also be referred to as a reagent for resolution.
Figure JPOXMLDOC01-appb-C000014
 (In the formula, R represents an arbitrary side chain of an amino acid)

　さらに別の態様では、本発明は、本発明の化合物を、アミノ酸と反応させることを含む、アミノ酸の蛍光誘導体を製造する方法にも関する。また当該製造方法により製造されたアミノ酸の蛍光誘導体、すなわち以下の式：

Figure JPOXMLDOC01-appb-C000015
（式中、Ｒは、任意のアミノ酸の側鎖を表す）
を有する、アミノ酸蛍光誘導体にも関する。本発明において、Ｒは、任意のアミノ酸の側鎖を表すが、タンパク質構成アミノ酸の側鎖であってもよい。さらに光学分割する観点では、タンパク質構成アミノ酸のうち、グリシン側鎖（-Ｈ）を除く任意の側鎖であることが好ましい。代表的には、アラニン側鎖（－ＣＨ3）又はトリプトファン側鎖（
Figure JPOXMLDOC01-appb-C000016
 ）がある。これらのアミノ酸蛍光誘導体は、ＮＢＤ部分に起因した蛍光を発することができ、また一部のアミノ酸側鎖の構造に起因して生じうる蛍光消光が生じない点に特徴がある。また、これらのアミノ酸蛍光誘導体は、元のアミノ酸の有するキラリティを保持している。したがって、アミノ酸蛍光誘導体をキラルカラムクロマトグラフィーに導入することで、元のアミノ酸のキラリティに基づき、Ｄ－アミノ酸とＬ－アミノ酸の分離が可能である点も特徴となる。 In yet another aspect, the present invention also relates to a method for producing a fluorescent derivative of an amino acid comprising reacting the compound of the present invention with an amino acid. Further, a fluorescent derivative of an amino acid produced by the production method, that is, the following formula:
Figure JPOXMLDOC01-appb-C000015
 (In the formula, R represents a side chain of any amino acid)
It also relates to amino acid fluorescent derivatives having In the present invention, R represents a side chain of any amino acid, but may be a side chain of a protein-constituting amino acid. Further, from the viewpoint of optical resolution, it is preferably an arbitrary side chain other than the glycine side chain (-H) among the amino acids constituting the protein. Typically, alanine side chain (—CH 3 ) or tryptophan side chain (
Figure JPOXMLDOC01-appb-C000016
 ) These amino acid fluorescent derivatives are characterized in that they can emit fluorescence due to the NBD moiety and do not cause fluorescence quenching that may occur due to the structure of some amino acid side chains. Further, these amino acid fluorescent derivatives retain the chirality of the original amino acid. Therefore, it is also a feature that D-amino acid and L-amino acid can be separated based on the chirality of the original amino acid by introducing an amino acid fluorescent derivative into chiral column chromatography.

　従来技術であるＮＢＤ－Ｆを用いて誘導体化された場合に、トリプトファン誘導体は、消光が生じていた（図１及び図２Ａ）。したがって、消光を生じないトリプトファン誘導体は、Ｄ－アミノ酸の網羅的解析の上で特に有用である。本発明の実施例において、上述のアミノ酸誘導体のうち、トリプトファン誘導体において、消光が生じないことが明らかにされた（図２Ｂ）。ここで、消光は、理論に限定されることを意図するものではないが、化合物中の蛍光性を有する構造に隣接する構造に起因して蛍光が失われることをいう。 When derivatized with NBD-F, which is a conventional technology, the tryptophan derivative was quenched (FIGS. 1 and 2A). Therefore, tryptophan derivatives that do not cause quenching are particularly useful for comprehensive analysis of D-amino acids. In the Example of this invention, it was clarified that quenching does not occur in tryptophan derivatives among the above-mentioned amino acid derivatives (FIG. 2B). Here, quenching is not intended to be limited to theory, but refers to loss of fluorescence due to a structure adjacent to a fluorescent structure in the compound.

　本発明の化合物を、光学異性体を有する化合物、例えばアミノ酸（Ｄ－アミノ酸＋Ｌ－アミノ酸）と反応させて得たアミノ酸の蛍光誘導体に対して、光学分割クロマトグラフィーを行うことにより、元の光学異性に基づいて、分離及び分析が可能になる。したがって、本発明のさらに別の態様では、本発明の化合物を含む、光学分割用試薬に関する。光学分割及び／又は蛍光誘導体化用試薬と、光学分割用カラムとを含む光学分割及び／又は蛍光誘導体化用のキット又はシステムに関していてもよい。 By performing optical resolution chromatography on a fluorescent derivative of an amino acid obtained by reacting the compound of the present invention with a compound having an optical isomer, for example, an amino acid (D-amino acid + L-amino acid), the original optical isomerism is obtained. Based on this, separation and analysis becomes possible. Accordingly, still another aspect of the present invention relates to an optical resolution reagent containing the compound of the present invention. It may be related to a kit or system for optical resolution and / or fluorescence derivatization including an optical resolution and / or fluorescence derivatization reagent and an optical resolution column.

　本発明の別の態様では、本発明は、試料中のアミノ基含有化合物を分析する方法に関する。具体的に、アミノ基含有化合物を含む試料と、以下の式：

Figure JPOXMLDOC01-appb-C000017
　で表される化合物とを反応させる工程；
　反応物をクロマトグラフィーに導入する工程
　を含む。本発明の分析方法は、特に光学異性体を分離する光学分割方法であることが好ましく、その場合クロマトグラフィーとしては、キラルクロマトグラフィーが行われる。アミノ基を有する化合物であれば、任意の化合物を用いることができる。例えばアミノ酸が挙げられ、アミノ酸、特にタンパク構成アミノ酸の光学異性体、すなわちＤ－アミノ酸とＬ－アミノ酸を分離して分析することができる。 In another aspect of the present invention, the present invention relates to a method for analyzing an amino group-containing compound in a sample. Specifically, a sample containing an amino group-containing compound and the following formula:
Figure JPOXMLDOC01-appb-C000017
 Reacting with a compound represented by:
Introducing the reactant into the chromatography. The analysis method of the present invention is particularly preferably an optical resolution method for separating optical isomers. In this case, chiral chromatography is performed as chromatography. Any compound having an amino group can be used. For example, amino acids can be mentioned, and optical isomers of amino acids, particularly protein-constituting amino acids, that is, D-amino acids and L-amino acids can be separated and analyzed.

　アミノ基含有化合物を含む試料と本発明の化合物とを反応させる工程は、アミノ基含有化合物を含む試料と、本発明の化合物を含む溶液とを混合し、場合により加熱することで反応を行うことができる。この反応は、蛍光退色を避ける目的から暗所で行われることが好ましい。適切な溶媒を任意に使用することができ、例えば水、メタノール、エタノール、アセトニトリルなどを用いることができる。 The step of reacting the sample containing the amino group-containing compound with the compound of the present invention comprises reacting the sample containing the amino group-containing compound with the solution containing the compound of the present invention and optionally heating. Can do. This reaction is preferably performed in the dark for the purpose of avoiding fluorescent fading. A suitable solvent can be arbitrarily used, for example, water, methanol, ethanol, acetonitrile and the like can be used.

　アミノ基含有化合物を含む試料は、生体試料、環境試料、実験試料、工業試料（食品試料、医薬品試料、化粧品試料）などの任意の試料であってよい。生体試料としては、血液、唾液、涙液、リンパ液などの体液、細胞や臓器などの組織試料、及び尿や糞便などの排泄物試料が挙げられる。これらの試料を直接、本発明の化合物と反応させてもよいが、反応工程の前に、試料に応じて適宜精製することが好ましい。また、反応工程後に精製を行うこともできる。試料の精製は、濾過、徐タンパク、溶媒抽出、イオン交換、固相抽出などが、試料の種類に応じて適宜選択して、または組み合わせて行うことができる。 The sample containing the amino group-containing compound may be any sample such as a biological sample, an environmental sample, an experimental sample, and an industrial sample (food sample, pharmaceutical sample, cosmetic sample). Biological samples include body fluids such as blood, saliva, tears and lymph, tissue samples such as cells and organs, and excrement samples such as urine and feces. These samples may be directly reacted with the compound of the present invention, but it is preferable to purify appropriately according to the sample before the reaction step. Further, purification can be performed after the reaction step. Purification of the sample can be performed by appropriately selecting or combining filtration, slow protein, solvent extraction, ion exchange, solid phase extraction, and the like according to the type of sample.

　上述の反応工程において、本発明の化合物と反応し、誘導体化されたアミノ基含有化合物は、光学分割カラムを備えたクロマトグラフィーに供される。クロマトグラフィーとしては、ＨＰＬＣが好ましい。光学分割カラムを備えたクロマトグラフィーに供する前に、別のカラム系を用いて予めクロマトグラフィーを行っていてもよい。光学分割カラムは、本発明の化合物により誘導体化されたＤ－アミノ酸とＬ－アミノ酸を分離できるカラムを任意に選択することができる。光学分割カラムは、一方の光学異性体のみを含む化合物を固定相として用いたものであり、例えば一例として、グリシン誘導体、ロイシン誘導体、キニーネ誘導体等を使用することができる。クロマトグラフィーによりＤ－アミノ酸とＬ－アミノ酸とに分離されたアミノ酸誘導体について、その蛍光を検出することにより分析することができる。蛍光の検出は、クロマトグラフィーシステムに含まれる検出器において行われうる。検出器は、ＮＢＤを励起する励起光（４７０ｎｍ）を照射し、励起したＮＢＤに由来する蛍光（蛍光波長：５３０ｎｍ）を検出することで、誘導体化されたアミノ酸を分析することができる。 In the reaction step described above, the amino group-containing compound that has been derivatized by reacting with the compound of the present invention is subjected to chromatography equipped with an optical resolution column. As chromatography, HPLC is preferred. Before being subjected to chromatography provided with an optical resolution column, chromatography may be performed in advance using another column system. As the optical resolution column, a column capable of separating D-amino acid and L-amino acid derivatized with the compound of the present invention can be arbitrarily selected. The optical resolution column uses a compound containing only one optical isomer as a stationary phase. For example, a glycine derivative, a leucine derivative, a quinine derivative, or the like can be used. The amino acid derivative separated into D-amino acid and L-amino acid by chromatography can be analyzed by detecting the fluorescence. The detection of fluorescence can be performed in a detector included in the chromatography system. The detector can analyze the derivatized amino acid by irradiating excitation light (470 nm) for exciting NBD and detecting fluorescence (fluorescence wavelength: 530 nm) derived from the excited NBD.

　タンパク質構成アミノ酸とは、タンパク質を構成する２０種類のアミノ酸を指す。タンパク質構成アミノ酸としては、アラニン（Ａｌａ／Ａ）、アルギニン（Ａｒｇ／Ｒ）、アスパラギン（Ａｓｎ／Ｎ）、アスパラギン酸（Ａｓｐ／Ｄ）、システイン（Ｃｙｓ／Ｃ）、グルタミン酸（Ｇｌｕ／Ｅ）、グルタミン（Ｇｌｎ／Ｑ）、グリシン（Ｇｌｙ／Ｇ）、ヒスチジン（Ｈｉｓ／Ｈ）、イソロイシン（Ｉｌｅ／Ｉ）、ロイシン（Ｌｅｕ／Ｌ）、リシン（Ｌｙｓ／Ｋ）、メチオニン（Ｍｅｔ／Ｍ）、フェニルアラニン（Ｐｈｅ／Ｆ）、プロリン（Ｐｒｏ／Ｐ）、セリン（Ｓｅｒ／Ｓ）、トレオニン（Ｔｈｒ／Ｔ）、トリプトファン（Ｔｒｐ／Ｗ）、チロシン（Ｔｙｒ／Ｙ）、及びバリン（Ｖａｌ／Ｖ）が挙げられる。これらのアミノ酸のうち、グリシンを除くアミノ酸について、Ｄ－アミノ酸及びＬ－アミノ酸の光学異性体が存在する。また、スレオニンとイソロイシンにはアロ体が存在する。本発明の試料中のアミノ酸を光学分割して分析する方法において、分析されるアミノ酸は、好ましくはグリシンを除くタンパク質構成アミノ酸であるが、その他のアミノ酸、例えばオルニチン、シトルリン等についても分析することができる。 Protein-constituting amino acids refer to 20 types of amino acids that constitute proteins. As protein constituting amino acids, alanine (Ala / A), arginine (Arg / R), asparagine (Asn / N), aspartic acid (Asp / D), cysteine (Cys / C), glutamic acid (Glu / E), glutamine (Gln / Q), glycine (Gly / G), histidine (His / H), isoleucine (Ile / I), leucine (Leu / L), lysine (Lys / K), methionine (Met / M), phenylalanine ( Phe / F), proline (Pro / P), serine (Ser / S), threonine (Thr / T), tryptophan (Trp / W), tyrosine (Tyr / Y), and valine (Val / V). . Among these amino acids, optical isomers of D-amino acids and L-amino acids exist for amino acids other than glycine. In addition, allone exists in threonine and isoleucine. In the method for optically resolving and analyzing amino acids in the sample of the present invention, the amino acids to be analyzed are preferably protein-constituting amino acids excluding glycine, but other amino acids such as ornithine and citrulline can also be analyzed. it can.

実施例１：２，５－ジオキソピロリジン―１－イル２－[Ｎ‐（４－ニトロベンゾ[ｃ][１，２，５]オキサジアゾール―７－イル）‐Ｎ‐メチルアミノ]アセテート（ＮＢＤ－ＣＯＯＳｕ）の合成
（１）ＮＢＤ－ＣＯＯＨの合成

Figure JPOXMLDOC01-appb-C000018
　暗所にて、Ｎ－メチルグリシン（１．８５ｇ、２０．８ｍｍｏｌ）を０．５２Ｍ炭酸水素ナトリウム水溶液（１００ｍＬ）に溶解し、これにＮＢＤ－Ｃｌ（１．０４ｇ、５．２３ｍｍｏｌ）のアセトニトリル溶液（５０ｍＬ）を滴下して加えた。溶液を室温で２時間撹拌した後、減圧下でアセトニトリルを留去した。溶液に６Ｍ塩酸水溶液（１４ｍＬ）を加えて酸性とした後、室温で３０分間撹拌した。析出した固体を吸引濾過し、減圧下で乾燥して、橙色固体を得た（1.35 g, quant.）。
　1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J= 9.0Hz, 1H), 6.47 (d, J=9.2 Hz, 1H), 4.91 (s, 2H), 3.47 (s, 3H)。 Example 1: 2,5-Dioxopyrrolidin-1-yl 2- [N- (4-nitrobenzo [c] [1,2,5] oxadiazol-7-yl) -N-methylamino] acetate ( Synthesis of NBD-COOSu) (1) Synthesis of NBD-COOH
Figure JPOXMLDOC01-appb-C000018
 In the dark, N-methylglycine (1.85 g, 20.8 mmol) was dissolved in 0.52 M aqueous sodium hydrogen carbonate solution (100 mL), and NBD-Cl (1.04 g, 5.23 mmol) in acetonitrile was dissolved in the solution. (50 mL) was added dropwise. After stirring the solution at room temperature for 2 hours, acetonitrile was distilled off under reduced pressure. The solution was acidified with 6M aqueous hydrochloric acid (14 mL), and then stirred at room temperature for 30 minutes. The precipitated solid was filtered with suction and dried under reduced pressure to obtain an orange solid (1.35 g, quant.).
1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 9.0 Hz, 1H), 6.47 (d, J = 9.2 Hz, 1H), 4.91 (s, 2H), 3.47 (s, 3H).

（２）　２，５－ジオキソピロリジン―１－イル２－[Ｎ‐（４－ニトロベンゾ[ｃ][１，２，５]オキサジアゾール―７－イル）‐Ｎ‐メチルアミノ]アセテート（ＮＢＤ－ＣＯＯＳｕ）の合成

Figure JPOXMLDOC01-appb-C000019
　暗所にて、実施例１で調製したＮＢＤ－ＣＯＯＨ（２３６ｍｇ、０．９３５ｍｍｏｌ）を塩化メチレン（２ｍＬ）に溶解し、塩化オキサリル（１．１ｍＬ）を滴下して溶液に加えた。Ｎ，Ｎ－ジメチルホルムアミド（５μＬ）を溶液に加え、室温で１時間撹拌した。その後、減圧下で溶媒を留去し、真空乾燥した。得られた残渣を塩化メチレン（１ｍＬ）に溶解し、Ｎ－ヒドロキシコハク酸イミド（１１５．２ｍｇ、１．００ｍｍｏｌ）及びＮ－メチルモルホリン（０．１ｍＬ）を塩化メチレン（１ｍＬ）に溶解した溶液に滴下した。１日の撹拌後、塩化メチレン（５０ｍＬ）を加え、水（２０ｍＬ）で２回、飽和炭酸水素ナトリウム水溶液（２０ｍＬ）で２回、水（２０ｍＬ）で１回洗浄し、硫酸ナトリウムで乾燥させた。硫酸ナトリウムをろ過した後、減圧下で溶媒を留去し、橙色の粉末を得た（２０６ｍｇ、収率６３．２％）
1H NMR (500 MHz, CD2Cl2) δ 8.50 (d, J= 8.7Hz, 1H), 6.32 (d, J=8.8 Hz, 1H), 5.26 (s, 2H), 3.52 (s, 3H), 2.84 (s, 4H)。 (2) 2,5-Dioxopyrrolidin-1-yl 2- [N- (4-nitrobenzo [c] [1,2,5] oxadiazol-7-yl) -N-methylamino] acetate (NBD -COOSu)
Figure JPOXMLDOC01-appb-C000019
 In the dark, NBD-COOH prepared in Example 1 (236 mg, 0.935 mmol) was dissolved in methylene chloride (2 mL), and oxalyl chloride (1.1 mL) was added dropwise to the solution. N, N-dimethylformamide (5 μL) was added to the solution and stirred at room temperature for 1 hour. Thereafter, the solvent was distilled off under reduced pressure, and vacuum drying was performed. The obtained residue was dissolved in methylene chloride (1 mL), and N-hydroxysuccinimide (115.2 mg, 1.00 mmol) and N-methylmorpholine (0.1 mL) were dissolved in methylene chloride (1 mL). It was dripped. After stirring for 1 day, methylene chloride (50 mL) was added, washed twice with water (20 mL), twice with saturated aqueous sodium bicarbonate (20 mL), once with water (20 mL), and dried over sodium sulfate. . After filtering sodium sulfate, the solvent was distilled off under reduced pressure to obtain an orange powder (206 mg, yield 63.2%).
1H NMR (500 MHz, CD2Cl2) δ 8.50 (d, J = 8.7Hz, 1H), 6.32 (d, J = 8.8 Hz, 1H), 5.26 (s, 2H), 3.52 (s, 3H), 2.84 (s , 4H).

実施例２：ＮＢＤ－ＣＯＯＳｕによるアミノ酸の蛍光誘導体化

Figure JPOXMLDOC01-appb-C000020
Figure JPOXMLDOC01-appb-C000021
 　２０ｐＭのＤ－トリプトファン及び８０ｐＭのＬ－トリプトファンをそれぞれ含むトリプトファン水溶液（１０μＬ）、及び０．５ｐＭのＤ－アラニン及び２ｐＭのＬ－アラニンをそれぞれ含むアラニン水溶液（１０μＬ）に４００ｍＭホウ酸塩緩衝液（ｐＨ８．０）２０μＬを加え、さらに４０ｍＭのＮＢＤ－ＣＯＯＳｕのアセトニトリル溶液（５μＬ）加えて６０°Ｃで２分間反応させた。次に０．２％トリフルオロ酢酸水溶液（７５μＬ）を加えて反応を停止させて、アミノ酸誘導体（ＮＢＤ－ＣＯ－Ｔｒｐ、及びＮＢＤ－ＣＯ－Ａｌａ）を取得した。 Example 2: Fluorescence derivatization of amino acids with NBD-COOSu
Figure JPOXMLDOC01-appb-C000020
Figure JPOXMLDOC01-appb-C000021
 400 mM borate buffer solution (10 μL) containing 20 pM D-tryptophan and 80 pM L-tryptophan respectively, and 400 mM borate buffer solution (10 μL) containing 0.5 pM D-alanine and 2 pM L-alanine respectively (10 μL) 20 μL of pH 8.0) was added, and 40 mM NBD-COOSu in acetonitrile (5 μL) was added, followed by reaction at 60 ° C. for 2 minutes. Next, 0.2% aqueous trifluoroacetic acid solution (75 μL) was added to stop the reaction, and amino acid derivatives (NBD-CO-Trp and NBD-CO-Ala) were obtained.

実施例３：ＨＰＬＣによる分離
　実施例２において得られたアミノ酸誘導体を、ＨＰＬＣ分析に供した。ＨＰＬＣ分析に使用したカラムは、ＫＳＡＡＣＳＰ－００１Ｒ（１．５ｍｍ i.d.×２５０ｍｍ）をキラル固定相としたオリジナルＰｒｉｋｌｅ型カラム（特許５９７７７６５号）であり、移動相としては、アセトニトリル：メタノール（３０：７０）を用いた。検出は、蛍光（励起波長４７０ｎｍ、蛍光波長５３０ｎｍ）及びＵＶ／ＶＩＳ（測定波長４７０ｎｍ）検出器を用いて行った。アラニン誘導体水溶液を、ＨＰＬＣ分析に供した。分析後、カラムを洗い（メタノール１０分、ギ酸アンモウムで１０分、さらにメタノールで２０分）、溶出液で４０分間の安定化を行い、次いでトリプトファン誘導体水溶液をＨＰＬＣ分析に供した。蛍光検出により得られたクロマトグラムを図２Ｂに示す。また、蛍光検出に代えて、検出を質量分析により行った。質量分析により検出したクロマトグラムを図２Ｃに示す。 Example 3: Separation by HPLC The amino acid derivative obtained in Example 2 was subjected to HPLC analysis. The column used for the HPLC analysis is an original Prickle type column (Patent No. 597765) using KSAACSP-001R (1.5 mm id × 250 mm) as a chiral stationary phase, and acetonitrile: methanol (30:70) as a mobile phase. Was used. Detection was performed using a fluorescence (excitation wavelength: 470 nm, fluorescence wavelength: 530 nm) and UV / VIS (measurement wavelength: 470 nm) detector. The aqueous solution of alanine derivative was subjected to HPLC analysis. After the analysis, the column was washed (methanol 10 minutes, ammonium formate 10 minutes, and methanol 20 minutes), and the eluate was stabilized for 40 minutes, and then the tryptophan derivative aqueous solution was subjected to HPLC analysis. The chromatogram obtained by fluorescence detection is shown in FIG. 2B. Further, instead of fluorescence detection, detection was performed by mass spectrometry. The chromatogram detected by mass spectrometry is shown in FIG. 2C.

比較例１：ＮＢＤ－Ｆを用いた場合のＨＰＬＣ分析
　タンパク質構成アミノ酸及びアロ体を含むアミノ酸の標準試料に４００ｍＭホウ酸塩緩衝液（ｐＨ８．０）１０μＬを加え、さらに４０ｍＭのＮＢＤ－Ｆ溶液（５μＬ）加えて６０°Ｃで２分間反応させた。次に０．２％トリフルオロ酢酸水溶液（７５μＬ）を加えて反応を停止させて、アミノ酸誘導体を取得した。取得したアミノ酸誘導体を、４５℃に設定した逆相カラム（モノリシックＯＤＳカラム、０．５３ｍｍ　ｉ．ｄ．×１０００ｍｍ;資生堂）を備えたＨＰＬＣに供した。５～３５％ＭｅＣＮ、０～２０％ＴＨＦ、及び０．０５％ＴＦＡを含む水性移動相を用いて、移動相の流速を２５μＬ／分として勾配溶出を行った。蛍光検出により得られたクロマトグラムを図１に示す。トリプトファンの蛍光強度が際立って低く、定量が困難であることが示された。 Comparative Example 1: HPLC analysis using NBD-F 10 μL of 400 mM borate buffer (pH 8.0) was added to a standard sample of amino acids containing protein-constituting amino acids and allobodies, and a 40 mM NBD-F solution ( 5 μL) and reacted at 60 ° C. for 2 minutes. Next, 0.2% trifluoroacetic acid aqueous solution (75 μL) was added to stop the reaction to obtain an amino acid derivative. The obtained amino acid derivative was subjected to HPLC equipped with a reverse phase column (monolithic ODS column, 0.53 mm id × 1000 mm; Shiseido) set at 45 ° C. Gradient elution was performed using an aqueous mobile phase containing 5-35% MeCN, 0-20% THF, and 0.05% TFA with a mobile phase flow rate of 25 μL / min. A chromatogram obtained by fluorescence detection is shown in FIG. The fluorescence intensity of tryptophan was markedly low, indicating that quantification was difficult.

比較例２：ＮＢＤ－Ｆを用いた場合のキラルＨＰＬＣ分析
　２０ｐＭのＤ－トリプトファン及び８０ｐＭのＬ－トリプトファンをそれぞれ含むトリプトファン水溶液（１０μＬ）、及び０．５ｐＭのＤ－アラニン及び２ｐＭのＬ－アラニンをそれぞれ含むアラニン水溶液（１０μＬ）に４００ｍＭホウ酸塩緩衝液（ｐＨ８．０）１０μＬを加え、さらに４０ｍＭのＮＢＤ－Ｆ溶液（５μＬ）加えて６０°Ｃで２分間反応させた。次に０．２％トリフルオロ酢酸水溶液（７５μＬ）を加えて反応を停止させて、アミノ酸誘導体（ＮＢＤ－Ｔｒｐ、及びＮＢＤ－Ａｌａ）を取得した。
　取得したアミノ酸誘導体を、実施例３と同様にＨＰＬＣ分析に供した。蛍光検出により得られたクロマトグラムを図２Ａに示す。 Comparative Example 2: Chiral HPLC analysis using NBD-F. An aqueous tryptophan solution (10 μL) containing 20 pM D-tryptophan and 80 pM L-tryptophan, respectively, and 0.5 pM D-alanine and 2 pM L-alanine. 10 μL of 400 mM borate buffer (pH 8.0) was added to each alanine aqueous solution (10 μL), 40 mM NBD-F solution (5 μL) was further added, and the mixture was reacted at 60 ° C. for 2 minutes. Next, 0.2% aqueous trifluoroacetic acid solution (75 μL) was added to stop the reaction, and amino acid derivatives (NBD-Trp and NBD-Ala) were obtained.
The obtained amino acid derivative was subjected to HPLC analysis in the same manner as in Example 3. A chromatogram obtained by fluorescence detection is shown in FIG. 2A.

　比較例２において、従来技術であるＮＢＤ－Ｆを用いて、アラニン及びトリプトファンを誘導体化し、誘導体をそれぞれキラルＨＰＬＣに供した結果が示された。アラニンでは、Ｄ－アラニン及びＬ－アラニンのピークが分離して観察されたものの、トリプトファンではＤ－トリプトファン及びＬ－トリプトファンの両方のピークが極めて低く定量ができなかった（図２Ａ）。理論に限定されることを意図するものではないが、ＮＢＤ環と、トリプトファンのインドール環との間で、光誘起電子移動が生じた結果消光が生じたものと考えられる。一方で、実施例３において、ＮＢＤ－ＣＯＯＳｕを用いてアラニン及びトリプトファンを誘導体化し、誘導体をそれぞれキラルＨＰＬＣに供した結果が示された。アラニン及びトリプトファンの両方について、それぞれＤ体及びＬ体のピークが分離して観察された（図２Ｂ）。そしてこのピークは、質量分析で検出した結果と一致した。これらの結果から、ＮＢＤ－ＣＯＯＳｕを用いたアミノ酸の誘導体化は、Ｄ体及びＬ体を含むアミノ酸を分離して、検出するために有効であることが示された。 In Comparative Example 2, a result of derivatizing alanine and tryptophan using NBD-F, which is a conventional technique, and subjecting each derivative to chiral HPLC was shown. In alanine, D-alanine and L-alanine peaks were observed separately, but in tryptophan, both D-tryptophan and L-tryptophan peaks were extremely low and could not be quantified (FIG. 2A). While not intending to be limited by theory, it is believed that quenching occurred as a result of photoinduced electron transfer between the NBD ring and the indole ring of tryptophan. On the other hand, in Example 3, the results of derivatizing alanine and tryptophan using NBD-COOSu and subjecting each derivative to chiral HPLC were shown. For both alanine and tryptophan, D-form and L-form peaks were observed separately (FIG. 2B). This peak coincided with the result detected by mass spectrometry. From these results, it was shown that derivatization of amino acids using NBD-COOSu is effective for separating and detecting amino acids including D-form and L-form.

Claims (7)

　以下の式：
Figure JPOXMLDOC01-appb-C000001
 　で表される化合物、又はその塩。 The following formula:
Figure JPOXMLDOC01-appb-C000001
 Or a salt thereof. 　以下の式：
Figure JPOXMLDOC01-appb-C000002
 　で表される化合物、又はその塩を含む、蛍光誘導体化用試薬。 The following formula:
Figure JPOXMLDOC01-appb-C000002
 A reagent for fluorescent derivatization, comprising a compound represented by the formula: 　試料中のアミノ基含有化合物を分析する方法であって、
　アミノ基含有化合物を含む試料と、以下の式：
Figure JPOXMLDOC01-appb-C000003
 　で表される化合物とを反応させる工程；
　反応物をクロマトグラフィーに導入する工程
　を含む、前記方法。 A method for analyzing an amino group-containing compound in a sample, comprising:
A sample containing an amino group-containing compound and the following formula:
Figure JPOXMLDOC01-appb-C000003
 Reacting with a compound represented by:
Introducing the reactant into chromatography. 　前記分析方法が、光学異性体の光学分割方法であって、クロマトグラフィーがキラルクロマトグラフィーである、請求項３に記載の方法。 The method according to claim 3, wherein the analysis method is an optical resolution method of optical isomers, and the chromatography is chiral chromatography. 　前記アミノ基含有化合物が、アミノ酸である、請求項３又は４に記載の方法。 The method according to claim 3 or 4, wherein the amino group-containing compound is an amino acid. 　アミノ酸誘導体の製造方法であって、
　アミノ酸を、以下の式：
Figure JPOXMLDOC01-appb-C000004
 　で表される化合物と反応させることを含む、前記方法。 A method for producing an amino acid derivative, comprising:
Amino acids have the following formula:
Figure JPOXMLDOC01-appb-C000004
 And reacting with a compound represented by: 　以下の式：
Figure JPOXMLDOC01-appb-C000005
 [式中、Ｒは、アミノ酸の側鎖である]
　で表される化合物。 The following formula:
Figure JPOXMLDOC01-appb-C000005
 [Wherein R is an amino acid side chain]
A compound represented by
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